The mouse intestinal epithelium undergoes continuous renewal throughout life. Intraepithelial lymphocytes (IELs) represent a significant fraction of this epithelium and play an important role in intestinal mucosal barrier function. We have generated a germ-free transgenic mouse model to examine the effects of a genetically engineered proliferative abnormality in the principal epithelial cell lineage (enterocytes) on IEL census and on IEL-enterocytic cross-talk. SV40 large T antigen (TAgWt) or a mutant derivative (TAgK107/8) that does not bind pRB was expressed in small intestinal villus enterocytes under the control of elements from the intestinal fatty acid binding protein gene (Fabpi). Quantitative immunohistochemical and flow cytometric analyses of conventionally raised and germ-free FVB/N Fabpi-TAgWt, Fabpi-TAgK107/8, and nontransgenic mice disclosed that forced reentry of enterocytes into the cell cycle is accompanied by an influx of thymically educated αβ T cell receptor (TCR)+ CD4+ and αβ TCR+ CD8αβ+ IELs and a decrease in intestinally derived γδ TCR+ CDαα IELs. Real time quantitative reverse transcriptase-PCR studies of jejunal villus epithelium recovered from germ-free transgenic and normal mice by laser capture microdissection and γδ TCR+ jejunal IELs purified by flow cytometry disclosed that the proliferative abnormality is accompanied by decreased expression of enterocytic interleukin-7 as well as IEL interleukin-7Rα and transforming growth factor β3. The analysis also revealed that normal villus epithelium expresses Fms-like tyrosine kinase 3 (Flt3), a known regulator of hematopoietic stem cell proliferation and neuronal cell survival, and its ligand (Flt3L). Epithelial expression of this receptor and its ligand is reduced by the proliferative abnormality, whereas IEL expression of Flt3L remains constant. Together, these findings demonstrate that changes in the proliferative status of the intestinal epithelium affects maturation of γδ TCR+ IELs and produces an influx of αβ TCR+ IELs even in the absence of a microflora.